CN115901712A - A kind of optical fiber Raman photometer and its construction method and application - Google Patents

Abstract

The invention discloses a fiber optic Raman photometer, including a light source part; a scanning system, the core of which is based on a laser confocal unit, including a first filter, a second filter, a first reflector, a second reflector, and a scanner , the objective lens, when the excitation light is generated from the fiber laser, it enters the scanner through the first filter, the first reflector, the second filter, and the second reflector in turn, and then focuses through the objective lens; the detection system, which is based on multiple The signal transmission and collection of the mode fiber, the end of the fiber is tapered, the incident light enters the fiber after being focused by the objective lens, the excitation light is emitted from the tapered end of the fiber, and the excitation probe generates a Raman signal, which is then collected through the same fiber; signal collection system, the signal collection system partially overlaps with the scanning system. When the Raman signal collected by the optical fiber returns to the scanner, it returns to the Raman spectrometer through the second mirror and the second filter, and the Raman spectrometer reads out the Raman signal.

Description

A kind of optical fiber Raman photometer and its construction method and application

technical field

The invention belongs to the technical field of spectral imaging and biosensing, and relates to an optical fiber Raman photometer and its construction method and application.

Background technique

As the most advanced part of the central nervous system, changes in the internal environment of the brain will bring about a series of diseases. Especially in the deep brain region, the change of its internal environment has more important pathological significance. Carrying out research on deep brain regions is conducive to clarifying the structure and function of the nervous system, thereby revealing the neural mechanism of brain work, and providing a more reliable basis for the diagnosis and treatment of related diseases. Traditional functional magnetic resonance imaging and electrophysiological techniques are widely used in brain imaging analysis, but are generally limited by low spatial resolution and it is difficult to distinguish chemical signals of different substances. Based on the high spectral resolution of molecular fingerprint information, surface-enhanced Raman spectroscopy (SERS) provides an optimal strategy for detecting chemical signal changes of different substances, with the advantages of resistance to photobleaching and autofluorescence. However, in traditional Raman instruments, the penetration ability of excitation light is limited, and it can only be used for the detection of signals at the solution, cell or tissue level, and it is difficult to be used in vivo, especially for the collection of Raman signals in deep brain regions. Therefore, There is an urgent need to develop an instrument that can be used to collect Raman signals from deep brain regions in vivo.

Contents of the invention

The purpose of the present invention is to provide a construction method and application of a fiber optic Raman photometer. The fiber optic Raman photometer has good biocompatibility and can realize the acquisition of Raman signals in deep brain regions.

The present invention provides an optical fiber Raman photometer, as shown in FIG. 6 .

The fiber optic Raman photometer comprises the following components:

(1) Light source part. The light source part is a fiber laser. Its excitation center wavelength is 785nm±0.5nm, etc.; the output power is adjustable from 0-500mW; the line width is less than 0.1nm; the output interface is SMA905 or FC/PC; the working voltage is 220V.

(2) Scanning system. The core of the scanning system is based on the laser confocal unit, and its components include first filter, second filter, first mirror, second mirror, scanner, objective lens and other components. When the excitation light is generated from the laser, it enters the scanner through filters and mirrors, and then focuses through the objective lens. The first filter is a band-pass filter, which allows light of 785nm to pass through; the second filter is a notch filter, which is mainly used to filter incident light. Both the first reflection mirror and the second reflection mirror are total reflection mirrors. The scanner is a laser confocal scanning unit. The objective lens magnification is 10×, NA 0.25. As shown in Figure 7.

(3) Detection system. The detection system is mainly based on the signal transmission and collection of multimode optical fiber. The end of the optical fiber is tapered, the incident light enters the optical fiber after being focused by the objective lens, and the excitation light is emitted from the tapered end of the fiber to excite the probe to generate a Raman signal, which is then collected through the same optical fiber. The fiber is a multimode fiber with a core of 200 microns and a cladding of 25 microns. The numerical aperture (NA) is 0.22, and the transmission range is 400-1100nm. The fiber end taper length is 480 microns. As shown in Figure 8:

(4) Signal collection system. The signal collection system partially overlaps with the scanning system. When the Raman signal collected by the optical fiber returns to the scanner, it further passes through the second mirror and the second filter and returns to the Raman spectrometer, and the Raman spectrometer reads out the Raman signal. Its scanner is a laser confocal scanning unit. The objective lens magnification is 10×, NA 0.25. The second mirrors are total reflection mirrors; the second filter is a notch filter to filter 785nm incident light. As shown in Figure 9.

The invention provides a method for building a fiber optic Raman photometer, comprising the following steps:

Step 1: Assemble the confocal scanning unit, specifically including the following sub-steps:

Step 1-1: Install the first filter, the first mirror, the second filter, and the second mirror in sequence;

Step 1-2: Install the objective lens;

Step 2: Build a scanning system, including the following sub-steps:

Step 2-1: Connect the fiber laser to the confocal scanning unit through an optical fiber;

Step 2-2: Adjust the optical path, the test excitation light can pass through the confocal scanning unit and be transmitted by the objective lens;

Step 3: Build the detection system, including the following sub-steps:

Step 3-1: Taper the optical fiber to obtain a tapered optical fiber to improve the efficiency of optical fiber signal collection;

Step 3-2: Assemble the tapered optical fiber to the end of the scanning system, and couple the excitation light into the optical fiber;

Step 4: Build a signal collection system, including the following sub-steps:

Step 4-1: Connect the Raman spectrometer to the confocal scanning unit through an optical fiber;

Step 4-2: Test Raman signal collection.

The present invention also provides the application of the optical fiber Raman photometer constructed by the above method in collecting Raman signals in vivo and/or in vitro.

The present invention also provides the application of the optical fiber Raman photometer constructed by the method as described above in the collection of Raman molecular signals in vitro under the excitation of excitation light, and the Raman molecules are such as rhodamine B, copper cyanine, and cyanine dye 5 .

Wherein the wavelength of the excitation light is 633nm, 785nm, preferably 785nm.

Wherein, the Raman molecule concentration is 0.1-5mM, preferably 1mM.

Wherein, the scanning range is 100-3200cm ^-1 , preferably 1000-1800cm ^-1 .

The present invention also provides the application of the optical fiber Raman photometer constructed by the above method in the collection of Raman molecular signals in different brain regions under the excitation of excitation light, and the different brain regions are cortex, hippocampus, striatum, thalamus.

Wherein, the wavelength of the excitation light is 633nm, 785nm, preferably 785nm.

Wherein, the Raman molecule is rhodamine B, copper cyanine, cyanine dye 5; preferably, it is rhodamine B.

Wherein, the scanning range is 100-3200cm ^-1 , preferably 1000-1800cm ^-1 .

The beneficial effects of the present invention are: constructing an optical fiber Raman photometer, which can realize the acquisition of Raman signals in deep tissues of living animals, and this application function is not available in current commercial Raman instruments.

Description of drawings

FIG. 1 is a Raman spectrum diagram of a blank optical fiber detected at different excitation wavelengths in Example 1.

Fig. 2 is the Raman spectrum of (a) rhodamine B, (b) copper cyanine, and (c) cyanine 5 collected under 785nm wavelength laser excitation in Example 2.

Fig. 3 is a schematic diagram of different brain regions targeted by optical fibers in Example 3.

Fig. 4 is the Raman probe prepared in Example 3. (a) TEM characterization of gold nanostars; (b) particle size distribution of gold nanostars; (c) UV absorption characterization of gold nanostars (AuNS) and Raman probes (RhB@AuNS); (d) amino groups Raman spectra of rhodamine B (RhB) and Raman probe (RhB@AuNS) under 785nm excitation.

Fig. 5 is the Raman spectrum of the probe collected in different brain regions in Example 3. (a) cortex; (b) hippocampus; (c) striatum; (d) thalamus.

Fig. 6 is a schematic diagram of an optical fiber Raman photometer of the present invention.

Fig. 7 is a schematic diagram of a fiber optic Raman photometer scanning system of the present invention.

Fig. 8 is a schematic diagram of a fiber optic Raman photometer detection system of the present invention.

Fig. 9 is a schematic diagram of a fiber optic Raman photometer signal collection system of the present invention.

Detailed ways

In conjunction with the following specific embodiments and accompanying drawings, the invention will be further described in detail. The process, conditions, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge in this field, and the present invention has no special limitation content.

The invention discloses a construction method and application of an optical fiber Raman photometer. First, a portable Raman spectrum signal acquisition device was built. On this basis, a fiber optic Raman photometer was further built to achieve efficient collection of Raman signals in vitro and in vivo. The fiber optic Raman photometer consists of four parts, including a light source, a scanning system, a detection system, and a signal collection system. The invention also discloses the application of the optical fiber Raman system in collecting Raman signals in solutions and brain slices.

Example 1 Collection of Blank Optical Fiber Signals of Optical Fiber Raman Photometer

Figure 1 shows the Raman spectra of the optical fiber itself under the excitation of different excitation light by the optical fiber Raman photometer.

Example 2 Fiber optic Raman photometer is used to collect signals of different Raman molecules in solution

In order to evaluate the Raman signal collection of Raman molecules in solution by a fiber-optic Raman photometer, different molecules, including rhodamine B, copper cyanine, and cyanine dye 5, were dissolved in ethanol to prepare a 1 mM solution, and then the signal collect. The method for measuring Raman signal molecules in the solution in the present invention is simpler and more convenient than the existing instrument measurement method, and does not require an additional focusing process.

As shown in Figure 2, the Raman spectra of different Raman molecules have Raman characteristic peaks of different molecules.

Example 3 Fiber optic Raman photometer is used to collect Raman signals in different brain regions

To evaluate the use of fiber optic Raman photometers for the collection of Raman signals from different brain regions, the ability of fibers to target brain regions was first assessed. As shown in Figure 3, optical fibers can effectively target brain regions such as the cerebral cortex, hippocampus, striatum, and thalamus. On the other hand, to enhance the Raman signal of the molecule, gold nanostars (AuNS) were further synthesized. As shown in Figure 4, the transmission electron microscope results show that the prepared gold nanostars are uniformly dispersed, and the particle size is about 41.97±8.17nm. Subsequently, the aminorhodamine B molecule (RhB) was modified on the gold nanostars through the interaction between gold and amino groups. The ultraviolet absorption showed that the absorption of AuNS alone was around 760nm. When RhB was modified on AuNS, the absorption of AuNS occurred at 7nm red. shift, and an obvious RhB absorption peak (~550nm) appeared. In addition, Raman spectroscopy showed that when RhB molecules were modified to AuNS, the Raman signal of RhB was significantly enhanced. These results indicated that the RhB@AuNS probe was successfully prepared.

Subsequently, the RhB@AuNS probe was incubated with the brain slices for 30 minutes, and then the Raman signals of different brain regions were collected with a fiber optic photometer. As shown in Figure 5, the fiber optic Raman photometer can be used to collect Raman signals of probes in different brain regions. This is the first reported device that can collect Raman signals in deep brain regions.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (8)

1. A fiber optic Raman photometer, characterized in that, comprising: A light source part, the light source part is a fiber laser; Scanning system, the core of the scanning system is based on the laser confocal unit, including a first filter, a second filter, a first mirror, a second mirror, a scanner, and an objective lens. When the excitation light is generated from the fiber laser, the Enter the scanner through the first filter, the first mirror, the second filter, and the second mirror, and then focus through the objective lens; A detection system, the detection system is based on multimode optical fiber signal transmission and collection, the end of the optical fiber is tapered, the incident light enters the optical fiber after being focused by the objective lens, the excitation light is emitted from the tapered end of the optical fiber, and the excitation probe generates a Raman signal, Then collect through the same fiber; The signal collection system partially overlaps with the scanning system. When the Raman signal collected by the optical fiber returns to the scanner, it returns to the Raman spectrometer through the second mirror and the second filter, and the Raman spectrometer reads out the Raman signal. 2. The fiber optic Raman photometer according to claim 1, wherein, in the light source part, the excitation center wavelength of the laser is 785nm ± 0.5nm; the output power is adjustable from 0-500mW; the line width is less than 0.1nm ; The output interface is SMA905 or FC/PC; the working voltage is 220V. 3. fiber optic Raman photometer as claimed in claim 1, is characterized in that, in described scanning system, described first filter is band-pass filter, can allow the light of 785nm to pass through; The second filter is A notch filter for filtering 785nm incident light; the first mirror and the second mirror are total reflection mirrors, the scanner is a laser confocal scanning unit, and the magnification of the objective lens is 10×, NA 0.25. 4. fiber optic Raman photometer as claimed in claim 1, is characterized in that, in described detection system, described optical fiber is multimode optical fiber, and fiber core is 200 microns, and cladding is 25 microns, and numerical aperture (NA) is 0.22, the transmission range is 400-1100nm, and the vertebral length at the end of the optical fiber is 480 microns. 5. A method for building the fiber optic Raman photometer according to any one of claims 1-4, characterized in that, comprising the steps of: Step 1: Assemble the confocal scanning unit, specifically including the following sub-steps: Step 1-1: Install the first filter, the first mirror, the second filter, and the second mirror in sequence; Step 1-2: Install the objective lens; Step 2: Build a scanning system, including the following sub-steps: Step 2-1: Connect the fiber laser to the confocal scanning unit through an optical fiber; Step 2-2: Adjust the optical path, the test excitation light can pass through the confocal scanning unit and be transmitted by the objective lens; Step 3: Build the detection system, including the following sub-steps: Step 3-1: Taper the optical fiber to obtain a tapered optical fiber to improve the efficiency of optical fiber signal collection; Step 3-2: Assemble the tapered optical fiber to the end of the scanning system, and couple the excitation light into the optical fiber; Step 4: Build a signal collection system, including the following sub-steps: Step 4-1: Connect the Raman spectrometer to the confocal scanning unit through an optical fiber; Step 4-2: Test Raman signal collection. 6. The application of the optical fiber Raman photometer built by the method according to claim 5 in collecting Raman signals in vivo and/or in vitro. 7. The fiber optic Raman photometer that a kind of method as claimed in claim 5 builds is under excitation light excitation, the application in the collection of in vitro Raman molecular signal; Wherein, The Raman molecules include rhodamine B, copper cyanine, and cyanine dye 5; The excitation light includes 633nm and 785nm; The Raman molecule concentration is 0.1-5mM; The scanning range is 100-3200cm ^-1 . 8. The fiber optic Raman photometer built by the method as claimed in claim 5 is excited by the excitation light, and the application in the collection of Raman molecular signals in different brain regions; wherein, The different brain areas are cortex, hippocampus, striatum, and thalamus; The excitation light includes 633nm and 785nm; The Raman molecules include rhodamine B, copper cyanine, and cyanine dye 5; The scanning range is 100-3200cm ^-1 .

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